Escherichia coli was the host organism for production of the first approved recombinant protein therapeutic in 1982. We now know that most therapeutic proteins require N-linked protein glycosylation to achieve their full clinical efficacy. Since E. coli has not been capable of protein glycosylation, the majority of approved therapeutic proteins are now expressed in mammalian host cells. While mammalian cells can express N-linked glycoproteins, they can have several drawbacks including: (i) slow growth, (ii) expensive media, (iii) long development timelines, (iv) low volumetric productivity, (v) susceptibility to viral contamination, and (vi) product heterogeneity. This problem has not gone unnoticed by the scientific community, and several eukaryotic organisms have been re-engineered for expression of therapeutic glycoproteins. Unfortunately, all eukaryotic hosts - including Chinese hamster ovary cells, plant cells, insect cells, or even genetically engineered yeast - introduce nonhuman glycoforms that arise from native glycosylation pathways. Glycobia specializes in glycoengineering bacteria as a platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis of these proposed studies is that glycoengineered E. coli can be used to express therapeutic glycoproteins. In Phase I of this project, we engineered E. coli capable of glycosylating proteins with the eukaryotic core glycan (Man3GlcNAc2) that is the predominant glycan in both plant and insect cells. In Phase II of this project, we propose to further engineer E. coli to enable glycosylation of therapeutic proteins with terminally sialylated human glycans. Specifically, we propose to engineer E. coli to glycosylate therapeutic proteins with eukaryotic N-glycans by screening enzymes to: (i) preferentially glycosylate N-X-S/T glycosylation motifs and (ii) efficiently glycosylate therapeutic target proteins with eukaryotic glycans. Further, we propose to engineer E. coli to synthesize and transfer complex terminally sialylated N-glycans by: (i) extending the Man3GlcNAc2 biosynthetic pathway for the biosynthesis of terminally sialylated glycans and (ii) screening enzymes for their ability to transfer the complex human N-glycan to target proteins. The benchmark of success for this project is expression of a commercial glycoprotein in E. coli. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoproteins for both companies and patients. PUBLIC HEALTH RELEVANCE: Most approved therapeutic proteins require posttranslational N-linked protein glycosylation and, as a consequence, are expressed in eukaryotic host cells that can be expensive, susceptible to viral contamination, and prone to product heterogeneity. The outcomes are low profit margins for biotechnology and pharmaceutical companies and prices that are prohibitive to the healthcare consumer. The proposed studies focus on expressing safe, affordable, and controlled complex human glycoproteins in the simple bacterium Escherichia coli.